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Article

Pollen Morphological Inter- and Intraspecific Variability in Selected Species of Rubus L. (Rosaceae)

by
Kacper Lechowicz
1,*,
Jan Bocianowski
2 and
Dorota Wrońska-Pilarek
1
1
Department of Botany and Forest Habitats, Faculty of Forestry and Wood Technology, Poznań University of Life Sciences, Wojska Polskiego 71 d, 60-625 Poznań, Poland
2
Department of Mathematical and Statistical Methods, Faculty of Agronomy, Horticulture and Bioengineering, Poznań University of Life Sciences, Wojska Polskiego 28, 60-637 Poznań, Poland
*
Author to whom correspondence should be addressed.
Forests 2022, 13(11), 1946; https://doi.org/10.3390/f13111946
Submission received: 27 October 2022 / Revised: 14 November 2022 / Accepted: 15 November 2022 / Published: 18 November 2022
(This article belongs to the Section Forest Ecophysiology and Biology)
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Abstract

:
Pollen morphology is one of the important vehicles in resolving some taxonomic problems on the family, genera or species level and has become part of the many disciplines and collaborative approaches in plant systematics and evolution. Palynological studies on the large and taxonomically highly complex genus Rubus L. have been limited, particularly concerning pollen variability. The aim of current study was to investigate the scope of inter- and intraspecific variability of the studied species based on pollen morphology, as well as verify taxonomic usefulness of pollen traits in distinguishing the studied taxa. The sixteen native Poland and Europe Rubus species were studied. Over a dozen quantitative and qualitative features of 2100 pollen (70 samples of 30 pollen each) were analyzed. Exine ornamentation and pollen size proved to be most useful pollen traits in the assessment of variability in the studied Rubus species. Pollen traits did not confirm the currently adopted taxonomical division of the genus Rubus into subgenera, sections and series. The greatest interspecific variability was found for traits P, E, Le and d, while variability was lowest in the case of Exp, Exe, P/E, Le/P, d/E, Exp/P and Exe/E. Intraspecific variability was determined by the following features ordered from those least to most variable: P, E, d, Le, d/E, Exp/P, Exp, P/E and Le/P.

1. Introduction

The genus Rubus L. is one of the most taxonomically complicated groups within the Rosaceae family with a worldwide distribution, including hundreds or even thousands of published species names and infrageneric taxa [1,2,3]. The number of species in the genus Rubus L. may vary from 429 to 750 or up to more than 1000 accepted species worldwide [4,5,6,7,8,9]. According to the latest data reported by Govaerts et al. [3] this genus comprises 1409 accepted species, of which from 250 to 300 species grow in Central and North-Western Europe [8]. In Poland, the occurrence of 108 native species from the genus Rubus has been confirmed so far [10,11]. Many blackberry species are found outside their natural distribution area as a result of their application in horticulture, while some have been introduced accidentally [12].
The difficulty of the genus Rubus is due to the large number of species with similar morphological characteristics and often small, local ranges of natural occurrence, as well as polyploid hybridization and apparently frequent facultative apomixis [13,14]. Therefore, among brambles pollen of only 48 European species had been characterized until 2019 [15,16,17,18,19,20,21,22,23,24]. These data have been supplemented by the comprehensive palynological studies by Xiong et al. [25], who examined 155 species and 13 varieties representing all the 12 subgenera of this genus, and by Lechowicz et al. [26], who described pollen grains of 58 Polish and European Rubus species.
The crucial features of bramble pollen grains include exine ornamentation (ornamentation type, width and orientation of striae and grooves), length of colpori, type of the bridge, costae colpi and the number and size of perforations [16,18,19,20,21,22,23,25,26,27,28,29,30,31,32,33,34,35,36,37,38]. In the opinion of Tomlik-Wyremblewska [22,32], pollen size and shape prove to be poor criteria in species identification.
The aim of this study was to investigate pollen inter- and intraspecific variability in 16 Rubus species native to Poland and Europe, representing three subgenera, two sections and subsections and seven series, based on morphological features of pollen grains. Currently, there are few palynological studies on the intraspecific variability of blackberries. Moreover, we tested the taxonomical value of the studied pollen features to assess whether these features may be used to distinguish among the analyzed taxa of the genus Rubus.

2. Material and Methods

The blossoms were collected in the herbarium of the Institute of Dendrology, Polish Academy of Sciences in Kórnik (KOR) and stored in the herbarium of the Department of Botany and Forest Habitats, the Poznań University of Life Sciences (PZNF).
The study was carried out on 70 samples of 16 native Rubus species (Table 1). A significant part of the plant material comes from Poland, while single specimens were collected in Austria, Bulgaria and Germany. A list of the samples analyzed with their affiliation to particular taxa is shown in Table 1.
The taxonomic classification of the studied taxa from the genus Rubus was adopted according to Zieliński [10], with further modifications by Kosiński et al. [11]. The verification of the taxa was made by Prof. Jerzy Zieliński (Institute of Dendrology, Polish Academy of Sciences in Kórnik), a batologist–taxonomist specializing in the genus Rubus.
Several, randomly selected inflorescences (flowers) were collected from 70 blackberry localities in four European countries (Table 2). Pollen grains were analyzed according to the method of Erdtman [39]. Inflorescences collected from the herbarium were placed in tubes and then centrifuged with glacial acetic acid. Grains were mixed with the acetolysis solution, which consisted of nine parts acetic anhydrite and one part concentrated sulfuric acid. The mixture was then heated to the boiling point and kept in the water bath for 2–3 min. Samples were centrifuged in the acetolysis mixture, washed with acetic acid and centrifuged again. The pollen grain samples were then mixed with 96% alcohol and centrifuged four times, with processed grains subsequently divided into two groups. One half of the processed sample was immersed in an alcohol-based solution of glycerin for the light microscopy (LM), while the other was placed in 96% ethyl alcohol in preparation for scanning electron microscopy (SEM). The SEM observations were carried out using a Zeiss Evo 40 microscope, while the size of the acetolyzed pollen grains were measured at 400x magnification using a Levenhuk D870T microscope equipped with a camera and software enabling accurate grain measurement. Measurements taken from 30 mature, randomly selected, properly developed pollen grains were made using LM, with 2100 pollen grains measured in total.
The pollen grains were analyzed for nine quantitative charactersistics: length of the polar axis (P) and equatorial diameter (E), length of the ectoaperture (Le), the distance between apices of two ectocolpi (d), thickness of the exine along the polar axis (Exp) as well as the P/E, Le/P, d/E and Exp/P ratios. The pollen shape classes (P/E ratio) were adopted according to the classification proposed by Erdtman (1952): oblatespheroidal (0.89–0.99), spheroidal (1.00), prolatespheroidal (1.01–1.14), subprolate (1.15–1.33) and prolate (1.34–2.00). In addition, the following qualitative characteristics were also determined: outline, shape, operculum structure and exine ornamentation.
Exine ornamentation types were identified based on the classification proposed by Ueda and Tomita [40]. The striate exine ornamentation types and subtypes were characterized by the height and width of grooves, width of striae, the number and diameter of perforations.
The palynological terminology adopted by Punt et al. [41] and Halbritter et al. [42].
The normality of distribution of the nine traits, e.g., P, Le, d, E, Exp, P/E, Le/P, d/E and Exp/P, was tested using Shapiro–Wilk’s normality test [43] to verify whether the analysis of variance (ANOVA) met the assumption that the ANOVA model residuals followed a normal distribution. The homogeneity of variance was tested using Bartlett’s test. Box’s M test tested multivariate normality and homogeneity of variance–covariance matrices. All the traits had a normal distribution. Multivariate analysis of variance (MANOVA) was performed based on the following model: Y = XT + E, where: Y is the (n × p)-dimensional matrix of observations, n is the number of all observations, p is the number of traits (in this study p = 9), X is the (n × k)-dimensional matrix of design, k is the number of pollen samples (in this study k = 70), T is the (k × p)-dimensional matrix of unknown effects, and E–the (n × p)-dimensional matrix of residuals. A one-way (pollen samples) MANOVA was performed. Next, one-way analysis of variance (ANOVA) was carried out to determine the effects of Rubus samples on the variability of P, Le, d, E, P/E and Le/P. The minimal, maximal and mean values as well as standard deviations of traits were calculated. Moreover, Fisher’s least significant differences (LSDs), at the 0.001 level, were calculated for individual traits and on this basis homogeneous groups were established. The relationships between the nine observed traits were estimated using Pearson’s linear correlation coefficients based on the means of Rubus species. Relationships of six observed traits were presented in a heatmap. The results were also analyzed using multivariate methods. A canonical variance analysis (CVA) was applied to present a multi-trait assessment of the similarity of the tested Rubus species in a lower number of dimensions with the least possible loss of information. The Mahalanobis distance was suggested as a measure of “polytrait” Rubus species similarity [44], the significance of which was verified by means of critical value Dα called “the least significant distance” [45]. Mahalanobis distances were calculated for all species samples. The differences between the analyzed species were verified by cluster analysis using the nearest neighbor method and Euclidean distances and presented as a dendrogram. The GenStat v. 18 statistical software package (VSN International) was used for the analyses.

3. Results

3.1. General Morphological Description of Pollen

The results of MANOVA indicated that all studied Rubus samples were significantly different when investigated in terms of all nine quantitative traits jointly (Wilk’s λ = 0.1687; F = 6.42; p < 0.0001). The analysis of variance for the nine quantitative traits [P (F69;2030 = 17.62), Le (F69;2030 = 12.47), d (F69;2030 = 13.24), E (F69;2030 = 15.41), Exp (F69;2030 = 6.79), P/E (F69;2030 = 6.70), Le/P (F69;2030 = 3.51), d/E (F69;2030 = 10.96) and Exp/P (F69;2030 = 8.64)] confirmed variability of the tested species at a significance level α = 0.001 (Table 3, Table 4 and Table 5). The mean values and standard deviations for the observed traits indicated high variability among the tested samples, for which significant differences were found in terms of all the analyzed morphological traits (Table 3, Table 4 and Table 5). The density plots of P, Exe and P/E by Rubus species samples are presented in Figure 1, Figure 2 and Figure 3, respectively. A description of pollen morphology of the Rubus species studied is given below and illustrated in electron micrographs (Figure 4, Figure 5 and Figure 6). Morphological observations for the other quantitative traits of pollen grains are shown in Table 3, Table 4 and Table 5.
Pollen grains of the Rubus species studied were monads, isopolar and tricolporate (Figure 4 and Figure 5). In accordance with the pollen size classification by Erdtman [46] on the basis of the length of the polar axis (P), analyzed pollen grains were small- (10–25 μm; 81.4%) or medium-sized (25.1–50 μm; 18.6%). Pollen grains marked a small range of mean values for trait P, ranging from 20.54 to 26.76 μm.
The average length of the polar axis (P) was 23.70 μm (16.56–32.75) μm (Table 3). The smallest mean P was found for the pollen of R. opacus (20.54 μm), while the largest—for R. radula (26.76 μm) (Table 3). In the R. opacus sample, all measured pollen grains were small at a narrow range of polar axis length (17.13–26.54 μm). In turn, the longest pollen grains were found in R. radula (20.36–30.87 μm).
The equatorial diameter (E) was 20.57 (12.81–28.32) μm. The shortest mean equatorial diameter was recorded in the pollen of R. divaricatus (16.81 μm), while the longest was found in R. caesius (22.97 μm, Table 4).
The outline in the polar view was mainly circular with obtuse apices, rarely elliptic, whereas in the equatorial view, the outline was mostly elliptic, less often circular (Figure 4 and Figure 5).
The mean P/E ratio was 1.16, ranging from 0.90 in R. bifrons to 1.65 in R. henrici-egonis (Table 4). On average the P/E ratio values were always above 1.00, ranging from 1.04 in R. plicatus to 1.26 in R. divaricatus. Pollen grains of the examined species were most frequently subprolate (42.9%–901 pollen grains) or prolatespheroidal (42.5%–893), rarely prolate (7.9%–166) and oblatespheroidal (6.7%–140).
The mean exine thickness was 1.09 μm (Exp) (with a range of 0.50–2.00 μm). The relative thickness of the exine (Exp/P ratio) averaged 0.05 (Exp/P—from 0.02 to 0.10).
The polar area index (PAI) or apocolpium index, i.e., the d/E ratio, averaged 0.14 (0.04–0.33). The lowest mean value of this ratio (0.04) was recorded in R. bifrons, while the highest (0.33)–in R. caesius.
Pollen grains usually have three apertures–colpori. The colpori were arranged meridionally, regularly, more or less evenly spaced and they were usually long, with a mean length of 20.04 (12.85–29.08) µm (Table 3). On average, the shortest colpori were found in R. opacus and R. divaricatus (17.07 μm), while the longest was recorded in R. radula (23.15 μm). On average, the length of the colporus (Le) constituted 85% of the polar axis length (P) and ranged from 78 to 89%. The colpori were narrow, linear or fusiform in outline. Their width was variable and usually greatest in the equatorial region. Sculpturing of ectocolpus membranes approached rugulate, rarely partly psilate. Colpus margins frequently had small undulations (Figure 4). In all the studied species a bridge was observed, crossing the colpus at the equator and dividing it into two parts, formed by two bulges of the ectexine that meet in the middle (Figure 5). The bulges were of the same or unequal length.
Exine ornamentation in all the studied species was striate-perforate (Figure 6). Exine ornamentation elements were variable (Figure 6). Striae and grooves usually ran parallel to colpori and the polar axis, but frequently they also formed fingerprint-like twists. Striae were straight or forked and of varying length, width and height.
The investigated pollen of individual Rubus species was classified according to the striate exine ornamentation classification proposed by Ueda and Tomita [40]. The cited authors distinguished six types (I–VI) and six subtypes (I–III, each A and B). In our study only subtypes IIA, B and IIIA, B were found (Figure 6, Table 6). Two species (R. caesius–Figure 6b, R. idaeus–Figure 6c) belonged to the IIB subtype, which was characterized by fairly distinct striae with long gaps and frequently by prominent, numerous perforations. Subtype IIIB was represented by two species (R. constrictus, R. sulcatus–Figure 6d) which were characterized by having more distinct and long ridges than type II. Four species (R. bifrons, R. nessensis, R. opacus, R. saxatilis–Figure 6e) belonged to the IIIA subtype with higher (i.e., more conspicuous than in subtype IIB), fairly narrow striae in comparison with subtype IIB. Subtype IIA was represented by eight species (R. divaricatus, R. gracilis, R. henrici-egonis, R. plicatus–Figure 6a, R. praecox, R. radula, R. scissus, R. sprengelii) characterized by having prominent perforations and short intervals between ridges.
In all of the species (16), elliptic or circular perforations of different diameters (0.06–0.5 μm) were found at the bottom of the grooves (Figure 6). In the majority of the species studied the perforations were small, with similar diameters (0.1–0.2 μm) and they were more or less numerous.
Significant positive relationships were observed between P and Le (0.964), P and d (0.284), P and E (0.780), Le and d (0.254), Le and E (0.764), d and E (0.546), d and d/E (0.941) as well as Exp and Exp/P (0.749). Negative correlations were observed between d and P/E (−0.464), E and P/E (−0.502), Exp and Le/P (−0.284), P/E and d/E (−0.334), P and Exp/P (−0.575), Le and Exp/P (−0.607), d and Exp/P (−0.268), as well as E and Exp/P (−0.472) (Figure 7).
Individual traits were of varying importance and had different shares in the joint multivariate variation in the studied species. Analysis of the first two canonical variates for 70 Rubus species regarding the nine quantitative traits is shown in Figure 8. In the graph the coordinates of the point for a particular genotype were the values for the first and second canonical variate, respectively. The first two canonical variates accounted for 58.40% of the total variability between the individual species (Figure 8, Table 7). The most significant positive, linear relationship with the first canonical variate was found for Exp/P, while the negative–for P, Le, d and E (Table 7). The second canonical variate was significantly positively correlated with d and d/E, whereas it was negatively correlated with P, Exp and P/E (Table 7).
The greatest variation in terms of all the nine traits jointly measured with Mahalanobis distances was found for R. opacus 1 and R. radula 1 (distance between them amounted to 4.343). The greatest similarity was found between R. scissus 1 and R. sulcatus 3 (0.395) (data not shown).
In the dendrogram presented in Figure 9 all the examined Rubus species were divided into four groups as a result of agglomeration grouping using the Euclidean distance method. The first (I) group comprised 15 Rubus samples: R. caesius 1, R. divaricatus (samples 1–4), R. gracilis 4, R. henrici-egonis (samples 1, 3–5), R. idaeus 4, R. nessensis 1, R. opacus 1, R. plicatus 1 and R. sulcatus 2 (Figure 9). The second (II) group comprised two samples: R. bifrons 3 and R. radula 1; while group III was composed of six samples: R. nessensis 5, R. opacus 2, R. praecox 1, R. radula 4, R. sprengelii 1 and R. sulcatus 4. Group IV comprised the other 47 Rubus samples (Figure 9).

3.2. Interspecific Variability of Pollen Grains

Taking into consideration mean coefficients of variation, the analyzed Rubus species can be arranged as follows (from the least to the most variable): R. henrici-egonis, R. nessensis, R. radula, R. sulcatus, R. opacus, R. plicatus, R. gracilis, R. idaeus, R. caesius, R. constrictus, R. divaricatus, R. praecox, R. saxatilis, R. bifrons, R. scissus and R. sprengelii (Table 3, Table 4 and Table 5).
The greatest interspecific variability was found for R. henrici-egonis, R. nessensis, R. radula, R. sulcatus, R. opacus, R. plicatus, R. gracilis and R. idaeus. This variability was determined to the greatest degree by the following traits: P (F = 33.66; p < 0.001), d (F = 28.93; p < 0.001), d/E (F = 26.71; p < 0.001), Le (F = 25.45; p < 0.001) and E (F = 25.17; p < 0.001). In contrast, the lowest variability was recorded for R. sprengelii, R. scissus, R. bifrons, R. saxatilis, R. praecox, R. divaricatus, R. constrictus and R. caesius based on the following features: Exp (F = 1.79; p = 0.03), Le/P (F = 2.04; p = 0.01), Exp/P (F = 11.56; p < 0.001) and P/E (F = 13.25; p < 0.001) (Table 3, Table 4 and Table 5).

3.3. Intraspecific Variability of Pollen Grains

The dendrogram (Figure 9) showed clustering of samples of individual species with similar pollen grain characteristics into four groups. Group I is characterized by a considerable intraspecific similarity in relation to d, Le/P, Exp/P and d/E in R. divaricatus (samples 1–4), while for Le/P, P/E, Exp and d/E in R. henrici-egonis (samples 1, 3 and 4) (Table 8). No similarities were observed in blackberries of groups II and III. In turn, in group IV the greatest similarity was recorded for d, d/E, Exp and P/E in R. saxatilis (samples 1–5), followed by Le/P, P/E, d and E in R. praecox (samples 1–4), Le/P, E, Exp/P and Exp in R. scissus (samples 1–3) and Le/P, Le, P and P/E in R. constrictus (samples 1 and 2). The greatest intraspecific variability was found in R. nessensis (determined mainly byExp/P, Exp, P, E and Le), R. sulcatus (E, Exp, Exp/P, P/E and P), R. gracilis (E, P, Le, P/E and Exp/P), R. idaeus (P, E, d, Exp and Exp/P) and R. caesius (E, P, d, Le and d/E) (Table 8).

4. Discussion

Rosaceae Juss. is a large family consisting of 109 accepted genera [47], which taxonomy is highly complex, particularly in the case of the genus Rubus L. [48,49]. Traits of pollen grains due to their conservative character may be of great importance in investigations of taxonomic relationships in the case of such difficult genera as the large genus Rubus L. comprising 1409 accepted species [3], of which 108 grow in Poland [10,11].
In relation to studies on pollen morphological features, all palynologists agree that the most important pollen features for the species in the genus Rubus are related with exine ornamentation features such as width, number and course of grooves (muri) and the width of striae, as well as the number and diameter of perforations [22,23,25,26,32,33,35,40,50]. In a study by Li et al. [33] the 103 examined Rubus species from China belonged to four types of exine ornamentation (rugulate, striate, cerebroid and reticulate–perforate), which were further divided into 11 subtypes. Other palynologists distinguish in blackberries mainly striate or striate-perforate exine ornamentation [16,18,19,21,22,23,29,30,32,35,37,51]. Except for the typical striate ornamentation, also striate-scabrate, striate-rugulate or rugulate [22,32], echinate or gemmate [16], verrucate [16,29,30], baculate and clavate [18,19] or reticulate ornamentation [51] have been rarely observed. According to current palynological studies, European blackberry species are slightly less variable in terms of this feature than Asian ones. Our results confirm that study, because in the examined pollen grains, only one type of exine ornamentation (striate) was found. Ueda and Tomita [40] and Ueda [31] distinguished six types and six subtypes of striate exine ornamentation in species and other taxa from the genus Rosa and the family Rosaceae, including the genus Rubus. In our latest research [26] we classified into four types (types IV and VI were not identified) and five subtypes (I A, II A, B, III A, B). Our results were similar to findings of the cited authors, since most of the examined pollen belonged to the IIA and IIIA subtypes and no grains were found in the very rarely represented types IV and VI or subtype IB.
Several authors considered pollen size and shape as potentially important traits in the diagnosis of the analyzed Rubus species [21,23,51,52], but others claimed that they have no diagnostic importance [33,34]. Based on our results, we partially agree with the opinion of the former ones, because the length of the polar axis (P) proved to be an important feature, since on its basis pollen grains were divided into small (81.4%) and medium-sized (18.6%).
In our study, the greatest interspecific variability was found in R. henrici-egonis, R. nessensis, R. radula, R. sulcatus, R. opacus, R. plicatus, R. gracilis and R. idaeus. It turned out that it was determined to the greatest degree by P, and to a slightly lesser extent also by d, d/E as well as Le and E. In turn, the smallest intraspecific variability was recorded in R. sprengelii, R. scissus, R. bifrons, R. saxatilis, R. praecox, R. divaricatus, R. constrictus and R. caesius. It was determined by other traits (Exp, Le/P, Exp/P and P/E). Very similar results were also reported by Lechowicz et al. [26], who investigated interspecific variability in 58 blackberry species. The greatest interspecific variability was determined by P, E, Le and d, while the smallest by Exp, Exe, P/E, Le/P, d/E, Exp/P and Exe/E. It needs to be added here that in the cited publication one sample was analyzed for each of the species, whereas in this study our team examined from two to five samples, which may have resulted in slight differences in the recorded results. The range of interspecific variability in the studied blackberries based on pollen traits is not dependent on their natural occurrence ranges. High variability was observed both in such very rare species as those having only several localities in Poland (R. henrici-egonis, R. opacus), those with larger ranges of occurrence (R. gracilis, R. nessensis, R. radula, R. sulcatus), as well as common species with extensive natural occurrence ranges in Poland and in Europe (R. plicatus, R. idaeus). The situation was similar in the group of species showing limited variability.
The least variable pollen traits in the investigated Rubus species included Le/P (1.61%), P/E (4.31%), P (6.01%) and Le (6.12%), whiled (18.64%), d/E (15.91%), Exp/P (9.27%) and Exp (7.43%) exhibited the greatest variability. Similar results were reported by Wrońska-Pilarek and Jagodziński [53] when analyzing the same pollen features in 16 species from the genus Rosa L. The ordering of the traits from the least to the most variable was as follows: P, E, P/E, Le, Exp and Exp/P.
Analyses of intraspecific variability presented in this paper provided diverse results. The greatest intraspecific variability was found in R. nessensis, R. sulcatus, R. gracilis, R. idaeus and R. caesius (Table 8), while it was smallest in R. divaricatus, R. saxatilis, R. scissus and R. constrictus based on traits ordered here from the least to the most variable: P > E > d > Le > d/E > Exp/P > Exp > P/E > Le/P. Identical results were recorded by Singh et al. [54] when analyzing pollen grain of nine species from the genus Rosa L.
Within this study it was also investigated whether pollen traits confirm the currently accepted taxonomical division of the genus Rubus into subgenera, sections, subsections and series, as presented in Table 1. The distribution of the analyzed species in the dendrogram (Figure 9) did not confirm this division. Group I comprised species from two different subgenera: Idaeobatus (R. idaeus) and Rubus (R. nessensis, R. divaricatus, R. opacus, R. plicatus, R. sulcatus, R. henrici-egonis, R. gracilis, R. caesius). In turn, group II consisted of species from the same subsection: Hiemales (R. bifrons, R. radula). In the group III, in which R. nessensis, R. opacus and R. sulcatus from subsection Rubus are found together with R. praecox, R. sprengelii and R. radula from subsection Hiemales. Group IV comprised many species from three different subgenera (Cylactis, Ideobatus and Rubus), while most species belonged to the largest subgenus Rubus (Figure 9). Additionally, it was not observed for different groups to be formed by species belonging to the same series. Identical conclusions were drawn by Xiong et al. [25] when analyzing pollen of species coming from all the 12 subgenera of the genus Rubus L. In view of a lack of other studies on the subject it may be assumed that morphological traits of pollen may not be considered useful in the taxonomy of the described genus. It is the opinion of the authors of this study that in pollen of species from the genus Rubus the absence of taxonomic dependencies may result from apomixis, defined as asexual reproduction without fertilization replacing sexual reproduction, thus reducing natural variability. Similar results in relation to representatives of the genus Rubus L. were reported by Wrońska-Pilarek [23,35] and Lechowicz et al. [26].

5. Conclusions

Pollen features, which proved to be most useful for the assessment of the intra- and interspecific variability of the studied Rubus species were the exine ornamentation features (width, number and course of grooves and the width of striae, as well as the number and diameter of perforations) and pollen size.
The range of the interspecific variability of the studied species ordered from the most variable to the least variable was as follows: R. henrici-egonis, R. nessensis, R. radula, R. sulcatus, R. opacus, R. plicatus, R. gracilis, R. idaeus, R. caesius, R. constrictus, R. divaricatus, R. praecox, R. saxatilis, R. bifrons, R. scissus and R. sprengelii. Such an ordering of species shows no relationships with the taxonomic division of the genus Rubus L. or with the ranges of natural occurrence of these species.
The greatest effect on intraspecific variability of the studied species was found for the most variable features, i.e., the distance between apices of two ectocolpi (d, d/E) and exine thickness (Exp/P, Exp), while it was determined to a lesser degree by pollen size (E, P, P/E) and the length of apertures (Le, Le/P).
Grouping of the investigated blackberry species in the dendrogram (Figure 9) based on pollen traits did not confirm the currently adopted taxonomic division of the genus Rubus into subgenera, sections and series.
Nevertheless, we believe it is worth expanding the research on the difficulted genus Rubus and conducting a full taxonomic review to answer the hypotheses fully.

Author Contributions

Conceptualization, D.W.-P. and K.L.; methodology, D.W.-P., K.L. and J.B.; software, J.B.; validation, J.B.; formal analysis, D.W.-P., K.L. and J.B.; investigation, D.W.-P., K.L. and J.B.; resources, D.W.-P.; data curation, J.B.; writing—original draft preparation, D.W.-P. and K.L.; writing—review and editing, D.W.-P., K.L. and J.B.; visualization, K.L. and J.B.; supervision, D.W.-P. and K.L.; project administration, D.W.-P. and K.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article.

Acknowledgments

We kindly thank Anna Binczarowska (English language proof-reader) for linguistic support.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Forests 13 01946 g001 550
Figure 1. The density plot of P trait for Rubus species. The point “•” indicates trait observation for individual species.
Figure 1. The density plot of P trait for Rubus species. The point “•” indicates trait observation for individual species.
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Figure 2. The density plot of Exe trait for Rubus species. The point “•” indicates trait observation for individual species.
Figure 2. The density plot of Exe trait for Rubus species. The point “•” indicates trait observation for individual species.
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Forests 13 01946 g003 550
Figure 3. The density plot of P/E trait for Rubus species. The point “•” indicates trait observation for individual species.
Figure 3. The density plot of P/E trait for Rubus species. The point “•” indicates trait observation for individual species.
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Forests 13 01946 g004 550
Figure 4. Pollen grains of Rubus caesius, R. idaeus, R. henrici-egonis, R. plicatus, R. radula and R. saxatilis in polar view, (a–f).
Figure 4. Pollen grains of Rubus caesius, R. idaeus, R. henrici-egonis, R. plicatus, R. radula and R. saxatilis in polar view, (a–f).
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Forests 13 01946 g005 550
Figure 5. Pollen grains of Rubus bifrons, R. idaeus, R. sulcatus, R. radula, R. nessensis and R. gracilis in equatorial view, (a–f).
Figure 5. Pollen grains of Rubus bifrons, R. idaeus, R. sulcatus, R. radula, R. nessensis and R. gracilis in equatorial view, (a–f).
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Forests 13 01946 g006 550
Figure 6. Striate exine ornamentation types according to Ueda and Tomita (1989); see Table 6. Rubus plicatus—IIA, R. caesius—IIB, R. idaeus—IIB, R. sulcatus—IIIB, R. saxatilis—IIIA, (a–e).
Figure 6. Striate exine ornamentation types according to Ueda and Tomita (1989); see Table 6. Rubus plicatus—IIA, R. caesius—IIB, R. idaeus—IIB, R. sulcatus—IIIB, R. saxatilis—IIIA, (a–e).
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Forests 13 01946 g007 550
Figure 7. Heatmap for Pearson’s correlation coefficients between observed traits (rcr = 0.232) for Rubus species. The heatmap shows a graphical representation of the correlation matrix between pairs of observed traits. Every element of the correlation matrix is presented by a darkened square indicating the value at that location, using another color or darkening density. P–length of the polar axis, Le–length of the ectoaperture, d–the distance between apices of two ectocolpi, E–equatorial diameter, P/E–length of the polar axis and equatorial diameter ratio, Le/P–the ratio of the length of the ectoaperture and length of the polar axis.
Figure 7. Heatmap for Pearson’s correlation coefficients between observed traits (rcr = 0.232) for Rubus species. The heatmap shows a graphical representation of the correlation matrix between pairs of observed traits. Every element of the correlation matrix is presented by a darkened square indicating the value at that location, using another color or darkening density. P–length of the polar axis, Le–length of the ectoaperture, d–the distance between apices of two ectocolpi, E–equatorial diameter, P/E–length of the polar axis and equatorial diameter ratio, Le/P–the ratio of the length of the ectoaperture and length of the polar axis.
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Forests 13 01946 g008 550
Figure 8. Distribution of 70 Rubus samples in the space of the first two canonical variables.
Figure 8. Distribution of 70 Rubus samples in the space of the first two canonical variables.
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Forests 13 01946 g009 550
Figure 9. Clustering (neighbor joining method) of Rubus species based on nine morphological traits. The length of the lines indicates the similarity/distance between two species or among two groups of species and stated hierarchical clusters.
Figure 9. Clustering (neighbor joining method) of Rubus species based on nine morphological traits. The length of the lines indicates the similarity/distance between two species or among two groups of species and stated hierarchical clusters.
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Table
Table 1. The taxonomic classification of the Rubus species studied.
Table 1. The taxonomic classification of the Rubus species studied.
No.SpeciesSubgenusSectionSubsectionSeries
1R. bifrons VestRubusRubusHiemalesDiscolores
2R. caesius L.RubusCaesii--
3R. constrictus P. J. Müll. & LefévreRubusRubusRubusRubus
4R. divaricatus P. J. Müll.RubusRubusRubusRubus
5R. gracilis J. Presl & C. PreslRubusRubusHiemalesRhamnifolii
6R. henrici-egonis HolubRubusRubusHiemalesDiscolores
7R. idaeus L.Idaeobatus---
8R. nessensis W. HallRubusRubusRubusNessenses
9R. opacus FockeRubusRubusRubusRubus
10R. plicatus Weihe & NeesRubusRubusRubusRubus
11R. praecox Bertol.RubusRubusHiemalesDiscolores
12R. radula WeiheRubusRubusHiemalesRadulae
13R. saxatilis L.Cylactis--Saxatiles
14R. scissus W. C. R. WatsonRubusRubusRubusNessenses
15R. sprengelii WeiheRubusRubusHiemalesSprengeliani
16R. sulcatus VestRubusRubusRubusRubus
Table
Table 2. List of proveniences of the Rubus species samples studied.
Table 2. List of proveniences of the Rubus species samples studied.
No.SpeciesLocalitiesGeographical CoordinatesCollector, Herbarium
1R. bifrons 1Poland, Kietrz50°04′46.7″ N, 18°00′07.6″ EKosiński, Tomaszewski, Zieliński; KOR
2R. bifrons 2Poland, Mszana Dolna49°40′51.9″ N, 20°04′35.4″ EBiałobok; KOR
3R. bifrons 3Poland, Braszowice50°33′15.3″ N, 16°47′53.2″ EKosiński; KOR
4R. bifrons 4Poland, Faliszówka49°38′39.5″ N, 21°36′30.1″ EOklejewicz; KOR
5R. bifrons 5Poland, Pomorzowice50°17′02.1″ N, 17°45′33.4″ ETomlik, Zieliński; KOR
6R. caesius 1Poland, Skomętno Wielkie53°51′00.0″ N, 22°37′29.9″ ETomlik; KOR
7R. caesius 2Poland, Wiewiórczyn51°36′24.9″ N, 19°07′23.9″ EStefanek; KOR
8R. caesius 3Poland, Pilchowice50°12′57.8″ N, 18°33′49.3″ ETomlik, Zieliński; KOR
9R. caesius 4Poland, Piła-Młyn53°30′52.0″ N, 17°53′17.0″ EStefanek; KOR
10R. caesius 5Poland, Dzierżysław50°02′51.6″ N, 17°58′09.1″ ETomlik, Zieliński; KOR
11R. constrictus 1Poland, Ząbkowice Śląskie50°35′21.4″ N, 16°48′40.2″ EKosiński; KOR
12R. constrictus 2Poland, Staniszów50°50′24.0″ N, 15°44′13.1″ EOklejewicz; KOR
13R. divaricatus 1Poland, Lubin51°23′49.1″ N, 16°12′20.0″ EZieliński; KOR
14R. divaricatus 2Poland, Moryń52°48′40.0″ N, 14°21′10.0″ EMaliński; KOR
15R. divaricatus 3Poland, Antonin51°30′59.1″ N, 17°51′16.9″ EZieliński; KOR
16R. divaricatus 4Germany, Wiednitz51°23′14.1″ N, 14°01′45.6″ EHans-Werner; KOR
17R. gracilis 1Poland, Pątnów51°08′47.3″ N, 18°37′29.3″ EZieliński; KOR
18R. gracilis 2Poland, Świdwin53°46′28.1″ N, 15°46′38.8″ EKosiński; KOR
19R. gracilis 3Poland, Kutyły50°34′51.5″ N, 22°15′23.5″ EOklejewicz; KOR
20R. gracilis 4Poland, Marcinów50°23′06.7″ N, 16°41′19.9″ EKosiński; KOR
21R. gracilis 5Poland, Starkowo51°58′37.8″ N, 16°18′35.7″ EZieliński; KOR
22R. henrici-egonis 1Poland, Ząbkowice Śląskie50°35′21.4″ N, 16°48′40.2″ EKosiński; KOR
23R. henrici-egonis 2Poland, Kłodzko50°26′17.8″ N, 16°39′16.7″ EKosiński; KOR
24R. henrici-egonis 3Poland, Bardo50°30′24.3″ N, 16°44′24.9″ EKosiński; KOR
25R. henrici-egonis 4Poland, Gorzyce49°57′30.6″ N, 18°23′45.4″ EKosiński, Tomaszewski, Zieliński; KOR
26R. henrici-egonis 5Poland, Szklary49°27′58.7″ N, 21°50′28.2″ EKosiński, Zieliński; KOR
27R. idaeus 1Poland, Pamiątkowo52°33′15.1″ N, 16°40′56.9″ ETomlik, KOR
28R. idaeus 2Poland, Kórnik52°14′57.4″ N, 17°05′24.5″ EZieliński, KOR
29R. idaeus 3Poland, Lubowidz53°07′18.0″ N, 19°50′26.9″ EAnisimowicz; KOR
30R. idaeus 4Poland, Pakosław51°37′00.0″ N, 17°03′26.8″ EKortus; KOR
31R. idaeus 5Poland, Wąbrzeźno53°16′46.7″ N, 18°56′52.0″ EStempola; KOR
32R. nessensis 1Poland, Kozielec53°15′09.3″ N, 18°13′51.2″ EKrawiec; KOR
33R. nessensis 2Poland, Wolsztyn52°07′02.1″ N, 16°06′45.6″ EPaczoski; KOR
34R. nessensis 3Poland, Prabuty53°45′21.6″ N, 19°12′16.0″ EBoratyński, Zieliński; KOR
35R. nessensis 4Poland, Wyspowo54°33′51.0″ N, 18°18′15.9″ EMarkowski; KOR
36R. nessensis 5Poland, Brodnica53°15′28.9″ N, 19°24′19.6″ EBoratyński, Zieliński; KOR
37R. opacus 1Poland, Poźrzadło52°18′30.0″ N, 15°15′30.0″ EZieliński; KOR
38R. opacus 2Poland, Starkowo51°58′37.8″ N, 16°18′35.7″ EZieliński; KOR
39R. plicatus 1Poland, Karnocice53°51′34.5″ N, 14°31′04.3″ EPiotrowska; KOR
40R. plicatus 2Poland, Sarbsk54°45′07.2″ N, 17°40′02.7″ EZieliński; KOR
41R. plicatus 3Poland, Karkonosze50°46′35.2″ N, 15°36′18.9″ EBoratyńscy; KOR
42R. plicatus 4Poland, Jastrzębia Góra54°49′49.6″ N, 18°18′15.9″ EPiotrowska; KOR
43R. plicatus 5Poland, Kamienna Góra50°47′00.3″ N, 16°01′59.2″ EBoratyński, Zieliński; KOR
44R. praecox 1Austria, Klausen-Leopoldsdorf48°06′27.0″ N, 16°01′52.7″ EKosiński; KOR
45R. praecox 2Germany, Berlin52°29′03.0″ N, 13°14′08.0″ EMackiewicz; KOR
46R. praecox 3Poland, Burkatów50°47′42.1″ N, 16°27′59.6″ EBoratyński; KOR
47R. praecox 4Bulgaria, Blagoevgrad42°00′29.9″ N, 23°05′28.5″ EBoratyński, Browicz, Zieliński; KOR
48R. radula 1Poland, Potakówka49°42′47.2″ N, 21°36′06.7″ EOklejewicz; KOR
49R. radula 2Poland, Boszkowo51°58′28.1″ N, 16°19′41.9″ EHantz; KOR
50R. radula 3Poland, Psary51°33′44.2″ N, 21°37′09.9″ EDanielewicz, Maliński, Zieliński; KOR
51R. radula 4Poland, Boguszyn51°56′36.5″ N, 16°28′16.4″ EDanielewicz, Maliński; KOR
52R. radula 5Poland, Postomino51°58′28.1″ N, 16°19′41.9″ EZieliński; KOR
53R. saxatilis 1Germany, Wald47°43′20.9″ N, 10°33′24.8″ EHoffmann, KOR
54R. saxatilis 2Poland, Promno52°27′03.0″ N, 17°14′42.9″ EBrowicz; KOR
55R. saxatilis 3Poland, Lublin51°15′00.8″ N, 22°34′19.3″ EFijałkowski; KOR
56R. saxatilis 4Poland, Błażejewo54°13′45.6″ N, 22°18′19.5″ EBugała; KOR
57R. saxatilis 5Poland, Bodzewko51°49′56.0″ N, 17°06′19.9″ EKaczmarek; KOR
58R. scissus 1Poland, Zielin54°14′47.9″ N, 17°05′58.9″ EZieliński, KOR
59R. scissus 2Poland, Słowiński National Park54°41′57.3″ N, 17°19′10.9″ EZieliński; KOR
60R. scissus 3Poland, Rudniki51°02′13.0″ N, 18°36′01.6″ EZieliński; KOR
61R. sprengelii 1Poland, Wolin53°50′36.0″ N, 14°36′57.1″ EZieliński; KOR
62R. sprengelii 2Poland, Ciechnowo53°51′34.1″ N, 15°44′20.9″ EZieliński; KOR
63R. sprengelii 3Poland, Słowieńsko53°51′56.9″ N, 15°38′24.7″ EZieliński; KOR
64R. sprengelii 4Poland, Kłoczewo53°57′56.2″ N, 14°36′57.1″ EBoratyńska, Dolatowska, Zieliński; KOR
65R. sprengelii 5Poland, Dobiegniew52°58′05.5″ N, 15°45′17.4″ EBoratyńska, Dolatowska, Zieliński; KOR
66R. sulcatus 1Poland, Bystrzyca Kłodzka50°17′48.7″ N, 16°39′07.3″ EKosiński; KOR
67R. sulcatus 2Poland, Dydnia49°41′15.6″ N, 22°10′16.8″ EOklejewicz; KOR
68R. sulcatus 3Poland, Poraż49°29′05.5″ N, 22°12′55.5″ EOklejewicz; KOR
69R. sulcatus 4Poland, Jaśliska49°26′29.9″ N, 21°48′08.5″ EKaczmarek; KOR
70R. sulcatus 5Poland, Słowik50°49′54.4″ N, 20°32′20.3″ EKaznowski; KOR
Table
Table 3. Mean, minimal and maximal values as well as standard deviations (s.d.) for the length of the polar axis (P), length of ectoaperture (Le) and distance between apices of two ectocolpi (d).
Table 3. Mean, minimal and maximal values as well as standard deviations (s.d.) for the length of the polar axis (P), length of ectoaperture (Le) and distance between apices of two ectocolpi (d).
TraitPLed
No.SpeciesMeanHomogeneous GroupsMinMaxs.d.MeanHomogeneous GroupsMinMaxs.d.MeanHomogeneous GroupsMinMaxs.d.
1R. bifrons_125.53a--e19.6930.332.20621.22c-j18.2825.972.0392.36t-zA140.679
2R. bifrons_224.84b-i22.1427.861.47420.89c-m17.9324.581.7092.968f-w25.20.807
3R. bifrons_326.69a22.3932.752.49522.98ab18.829.082.5112.842h-x1.54.130.702
4R. bifrons_424.78b-i20.2530.52.19621.09c-k17.4726.252.0832.58p-z1.45.20.923
5R. bifrons_525.33a-f22.0130.852.18521.31c-j16.6726.832.2352.531q-zA1.73.840.605
6R. caesius_121.89w-zABC19.0326.361.64318.26v-y14.6322.671.7362.347u-zA1.173.80.672
7R. caesius_226.15ab22.5328.321.46522.38a-c19.0724.271.4293.85bc2.355.80.964
8R. caesius_325.72a-c22.0229.331.93221.75a-d17.1826.522.3463.77b-d26.31.102
9R. caesius_425.58a-e23.3226.910.94521.03c-k18.0724.191.4744.553a3.046.220.829
10R. caesius_523.68g-v19.4526.751.8519.83g-v15.8723.152.0474.075ab2.85.350.664
11R. constrictus_123.89f-u19.0827.582.26420.1e-s15.1924.232.4013.283c-o1.961.079
12R. constrictus_223.46h-w19.6727.281.51719.71j-v14.7322.951.6572.734k-z1.44.940.886
13R. divaricatus_122.34u-zAB19.1724.741.36119.15p-y15.422.081.5131.834A130.464
14R. divaricatus_221.01BC17.5224.211.8417.7Y1420.361.8032.066zA1.243.60.573
15R. divaricatus_321.76x-zABC19.0423.891.44518.17v-y13.8820.51.7872.088yzA1.073.390.594
16R. divaricatus_421.72x-zABC18.9625.261.34918.19v-y14.9621.591.492.047zA14.020.678
17R. gracilis_125.63a-d21.6531.572.56321.65a-e17.4727.662.6363.098d-s1.966.31.064
18R. gracilis_223.7g-v20.6926.271.43120.33d-s17.4523.081.5872.581o-z1.5550.834
19R. gracilis_324.69b-j20.7427.861.94521.41b-g17.6225.492.2283f-u1.55.350.950
20R. gracilis_422.74o-z20.2825.191.22119.29m-y16.3721.851.2652.442r-zA1.4550.731
21R. gracilis_525.46a-f21.6329.792.16321.23c-j16.4725.932.3012.72k-z1.64.90.859
22R. henrici-egonis_122.67r-A19.4127.251.84618.84r-y14.7523.281.7882.266w-zA1.294.60.642
23R. henrici-egonis_224.42c-m19.2728.372.48820.87c-n15.1924.512.4483.722b-e2.35.50.930
24R. henrici-egonis_322.42t-zAB19.3127.412.16218.91q-y15.0626.362.4422.151x-zA1.33.20.406
25R. henrici-egonis_421.23zABC18.9724.471.35617.92w-y15.2220.851.3322.288v-zA0.944.80.864
26R. henrici-egonis_522.7q-zA19.9127.711.61419.26n-y15.823.931.5962.614o-z1.224.020.658
27R. idaeus_122.16v-zAB19.8925.611.51819.23o-y16.3423.221.8663.444b-j25.71.062
28R. idaeus_224.62b-l22.1926.721.28820.32d-s16.1422.881.5044.053ab3.085.060.534
29R. idaeus_322.81n-z18.0726.182.12319.15p-y12.8521.781.9923.076d-s2.054.60.671
30R. idaeus_421.39yzABC16.56242.29318.39t-y14.0221.232.1213.321c-n2.034.210.669
31R. idaeus_522.52t-zAB19.0726.171.58819.12p-y14.6322.891.6733.542b-h2.54.750.569
32R. nessensis_123.1k-x19.826.691.82719.6k-v16.4723.852.1352.549p-z14.980.828
33R. nessensis_224e-t19.6729.932.65120.26d-s16.425.822.5463.07d-s1.65.511.055
34R. nessensis_325.63a-d22.1931.452.48821.34c-i17.7426.262.1682.597o-z1.36.130.970
35R. nessensis_424.9b-h21.6230.232.07620.82c-o16.7225.642.0142.987f-v1.295.561.064
36R. nessensis_524.31c-o21.4126.821.53920.34d-s17.824.281.7692.44r-zA1.54.030.632
37R. opacus_120.54C17.1323.81.69717.73xy15.5820.91.3712.147x-zA1.52.860.335
38R. opacus_222.96m-y20.7326.541.39318.46t-y13.9322.661.8663.061e-t1.85.510.915
39R. plicatus_123.11j-x18.8426.51.91319.75i-v16.0523.341.8372.638m-z1.564.240.652
40R. plicatus_221.15ABC17262.17318.36u-y14.2323.122.22.833i-x1.774.540.758
41R. plicatus_324.29c-p21.2526.621.42519.74i-v17.1121.981.233.138d-r1.561.206
42R. plicatus_423.46h-w20.7527.91.58719.33l-x16.0522.241.3923.333c-m26.20.931
43R. plicatus_522.7q-zA18.8525.211.66619.3l-y16.5221.581.563.653b-f25.650.975
44R. praecox_124.29c-p19.9829.781.53320.7d-p16.5324.141.4592.811j-x1.245.731.002
45R. praecox_222.27v-zAB18.6825.141.57618.87r-y16.1121.41.4393.384b-l25.520.734
46R. praecox_323.04l-x2126.011.45319.78h-v17.2323.141.4272.7l-z1.874.970.673
47R. praecox_425.36a-f21.9729.662.27521.77a-d18.5626.12.1292.979f-v25.090.694
48R. radula_126.76a23.4330.752.22623.15a20.428.632.1542.428s-zA1.323.660.639
49R. radula_223.63g-v20.3625.981.74320.09e-s14.5222.911.9432.62n-z1.943.70.425
50R. radula_325.63a-d21.6630.872.26721.29c-j15.1526.362.5253.153c-q1.56.251.208
51R. radula_424.27c-q20.4727.721.82519.98f-u16.9122.941.5863.324c-m2.055.70.922
52R. radula_525.05b-g22.0427.211.43621.25c-j17.9524.221.8382.153x-zA1.45.50.844
53R. saxatilis_124.48c-m21.8129.011.81121.57a-f18.4625.61.6953.669b-f2.095.810.913
54R. saxatilis_224.12d-s20.9627.81.48720f-t15.4623.131.9173.421b-k2.045.440.892
55R. saxatilis_322.63s-zA18.4125.31.64319.24o-y14.5821.831.6593.345c-l1.995.540.865
56R. saxatilis_422.22v-zAB16.925.81.94218.8s-y15.0421.81.6643.544b-h25.290.954
57R. saxatilis_522.54s-zAB19.6327.911.73219.15p-y15.8623.41.7493.239c-p2.294.530.590
58R. scissus_122.73p-zA20.5327.891.62519.18p-y14.8122.421.6952.597o-z1.63.750.607
59R. scissus_223.29i-x20.7225.611.11219.8g-v17.2324.111.393.529b-i1.86.41.190
60R. scissus_324.36c-n20.228.342.18320.64d-p16.7325.141.9953.576b-g25.81.044
61R. sprengelii_124.8b-i21.2728.721.91921.15c-k18.2425.311.9613.222c-q25.610.867
62R. sprengelii_223.5g-v21.228.361.50919.75i-v16.6424.381.7452.774j-y1.750.683
63R. sprengelii_324.74b-i2228.651.97520.91c-l17.6525.832.1922.707l-z1.765.10.843
64R. sprengelii_424.22c-r21.0626.21.45720.52d-q15.4523.021.582.997f-u24.40.652
65R. sprengelii_523.58g-v19.926.431.6920.45d-r16.923.551.832.9g-w25.50.863
66R. sulcatus_124.34c-n20.3831.022.73420.41d-s16.825.212.2853.011f-u25.730.911
67R. sulcatus_222.46t-zAB17.2526.041.85919.31l-y14.3822.761.9312.696l-z23.990.598
68R. sulcatus_323.1k-x2028.152.219.55k-v1525.372.1612.78j-y1.54.330.799
69R. sulcatus_423.09k-x19.0727.312.23419.38l-w15.823.362.1482.927g-w1.514.80.979
70R. sulcatus_524.66b-k22.0227.721.4521.38b-h17.9925.21.6682.734k-z1.454.720.786
LSD0.0011.581 1.618 0.702
F-ANOVA17.62 *** 12.47 *** 13.24 ***
*** p < 0.001; in the column means followed by the same letters are not significantly different.
Table
Table 4. Mean, minimal and maximal values as well as standard deviations (s.d.) for equatorial diameter (E), exine thickness along the polar axis (Exp) and P/E.
Table 4. Mean, minimal and maximal values as well as standard deviations (s.d.) for equatorial diameter (E), exine thickness along the polar axis (Exp) and P/E.
TraitEExpP/E
No.SpeciesMeanHomogeneous GroupsMinMaxs.d.MeanHomogeneous GroupsMinMaxs.d.MeanHomogeneous GroupsMinMaxs.d.
1R. bifrons_121.4a-m16.2124.922.1671.058g-q0.71.50.1631.204a-l0.8951.5300.150
2R. bifrons_221.54a-k18.4425.21.7711.06g-q0.81.380.1411.159d-s1.0251.3900.091
3R. bifrons_322.36a-e18.4725.841.9561.165a-i11.7050.1671.196a-m1.0581.4040.087
4R. bifrons_421.35b-n16.3726.532.4191.073f-q11.210.0821.171a-r1.0091.4940.132
5R. bifrons_521.01c-q1724.231.7750.99l-q0.81.20.1091.213a-i0.9791.4500.134
6R. caesius_118.39x-z1420.821.4981.096e-q11.30.0931.198a-m1.0061.5960.136
7R. caesius_222.92ab19.0825.051.4711.15a-j0.81.50.2501.143g-t1.0441.3570.063
8R. caesius_322.97a18.6327.132.0071.071f-q11.30.1051.124i-u0.9801.3970.094
9R. caesius_421.77a-j19.5223.931.2011.067g-q0.81.440.1521.177a-q1.0681.3080.061
10R. caesius_522.22a-f17.824.551.4691.048h-q11.20.0641.068tu0.9261.2080.079
11R. constrictus_121.32b-n17.225.572.311.068g-q11.20.0711.128i-t0.9311.4330.121
12R. constrictus_220.18j-w15.9824.341.9831.131b-l11.430.1571.168b-r1.0291.3700.079
13R. divaricatus_118.93v-y14.9922.31.6741.225a-e0.81.530.2141.187a-n1.0291.5000.104
14R. divaricatus_216.81z12.8121.191.7681.032i-q11.20.0651.26a1.0551.5270.144
15R. divaricatus_319.27s-y15.4524.571.91.109c-p11.50.1381.139g-t0.9551.4560.130
16R. divaricatus_419.15t-y17.0823.031.5461.185a-h0.81.50.2271.138g-t0.9591.3690.078
17R. gracilis_122.08a-g16.5725.722.0031.08e-q0.81.30.1611.167b-s1.0001.4620.127
18R. gracilis_220.7f-t18.722.811.1861.053h-q0.820.2291.147f-t1.0161.3460.084
19R. gracilis_321.43a-l17.7924.52.0431.063g-q0.91.20.1031.157d-t1.0031.3660.089
20R. gracilis_418.23yz16.3920.621.1571.098e-q0.81.50.1871.252ab1.0741.4220.093
21R. gracilis_521.3c-n19.3825.091.341.169a-i0.991.520.1511.196a-m1.0381.3720.084
22R. henrici-egonis_118.64w-y16.3322.31.4141.057g-q0.91.230.0861.219a-h1.0541.5470.098
23R. henrici-egonis_222.53a-c16.3625.712.0881.135a-l11.40.1371.086r-u0.9851.2410.074
24R. henrici-egonis_319.13t-y14.8822.561.891.083e-q0.991.80.1501.179a-p1.0011.5460.130
25R. henrici-egonis_418.91v-y15.2123.091.8321.202a-g0.691.750.2601.128i-t0.9931.2600.075
26R. henrici-egonis_519.12t-y13.822.421.9770.993l-q0.751.20.0991.201a-l0.9561.6500.161
27R. idaeus_120.13k-w13.8823.472.830.982n-q0.81.210.1181.121k-u0.9701.4430.163
28R. idaeus_222.46a-d21.0824.10.861.181a-h0.920.2871.097n-u0.9531.1820.058
29R. idaeus_319.43q-y14.3522.181.8471.05h-q0.71.30.1681.183a-o0.9101.4770.146
30R. idaeus_419.51p-y13.8725.12.7861.141a-k0.81.350.1431.105n-u0.9091.2250.080
31R. idaeus_520.77e-s16.9825.11.5431.088e-q11.360.1161.088q-u0.9371.3870.095
32R. nessensis_119.00u-y14.4822.351.8331.135a-l0.871.4440.1711.223a-g1.0101.5950.121
33R. nessensis_220.11k-w16.7525.182.0661.042h-q0.91.20.0681.198a-m0.9681.4510.117
34R. nessensis_320.8e-s17.3927.072.1341.052h-q0.781.230.1241.235a-f1.0581.3810.076
35R. nessensis_420.63f-t16.3623.661.8541.046h-q0.721.440.1751.211a-k1.1041.4850.094
36R. nessensis_519.79m-y15.5123.641.931.277a0.91.50.2101.241a-d0.9881.5160.150
37R. opacus_116.88z14.1619.631.6970.966pq0.81.20.1241.223a-g1.0191.4520.111
38R. opacus_218.61w-y15.4821.561.4891.269ab0.91.50.2561.24a-e1.0301.4210.118
39R. plicatus_121.3c-n19.1324.981.6391.094e-q11.50.1161.087q-u0.9321.2640.075
40R. plicatus_218.97u-y15.12262.7861.222a-e0.781.750.3131.125i-u0.9201.3290.099
41R. plicatus_321.79a-j18.2924.51.7371.124b-n0.51.50.2951.118l-u0.9701.2870.076
42R. plicatus_421.25c-n17.0325.241.9731.128b-m0.51.630.3021.109m-u1.0021.4830.087
43R. plicatus_521.95a-h17.0225.611.7620.963q0.61.20.1221.036u0.9081.1650.064
44R. praecox_121.67a-k19.4927.441.741.236a-d0.781.620.2671.125i-u0.9841.2960.085
45R. praecox_219.53o-y15.5623.031.9730.982n-q0.81.30.1161.15e-t0.9711.4580.131
46R. praecox_320.44h-v15.9126.052.4141.029i-q0.81.20.1111.137g-t0.9941.4120.099
47R. praecox_421.9a-i16.0728.322.5141.027i-q0.91.20.0791.168b-r1.0011.5180.132
48R. radula_122.14a-g19.1925.071.7081.202a-g0.751.630.2811.212a-j1.0061.4040.101
49R. radula_220.58g-u16.5425.561.7530.963q0.81.20.1271.156d-t0.9601.5560.133
50R. radula_321.84a-i18.1626.071.8391.071f-q11.220.0731.177a-q1.0161.3900.099
51R. radula_421.11c-p16.8424.452.0241.247a-c0.91.90.2651.155d-t1.0011.3850.097
52R. radula_521.82a-i18.6525.251.8341.1d-q11.40.1241.154d-t0.9681.4250.103
53R. saxatilis_120.31i-v17.1122.311.1620.977o-q0.81.20.1071.207a-l1.0491.3850.085
54R. saxatilis_220.86d-s18.4123.341.2971.153a-i0.51.520.2661.16c-s0.9981.3250.091
55R. saxatilis_320.83e-s16.0823.151.9141.12c-o0.721.820.2281.093o-u0.9151.3650.111
56R. saxatilis_419.77n-y16.1523.441.7571.007j-q0.71.20.1481.13h-t0.9521.4560.127
57R. saxatilis_519.37r-y16.5421.71.4271.047h-q0.91.60.1441.168b-r0.9971.3430.104
58R. scissus_120.94c-r17.9925.582.151.033i-q0.851.250.1251.091p-u0.9551.2910.081
59R. scissus_221.68a-k19.0325.131.491.096e-q0.91.20.1001.077s-u0.9931.2150.057
60R. scissus_321.13c-o17.1525.91.9291.068g-q0.981.20.0741.159d-s0.9891.4800.116
61R. sprengelii_121.08c-p17.8624.541.7071.216a-f0.91.50.2231.181a-p1.0031.3650.096
62R. sprengelii_220.72f-t16.7326.342.0910.993l-q0.81.20.1291.141g-t1.0091.3220.094
63R. sprengelii_319.92l-x16.325.342.2271.149a-j0.991.40.1191.25a-c1.1161.4680.104
64R. sprengelii_421c-q16.8425.422.3440.997k-q0.71.30.1771.163b-s0.9651.3730.108
65R. sprengelii_520.93c-r19.123.761.2870.985m-q0.81.110.0951.127i-t1.0151.2270.066
66R. sulcatus_121.65a-k17.3825.912.3481.104c-q0.781.3330.1551.127i-t1.0021.3040.082
67R. sulcatus_218.37x-z14.5725.472.5241k-q0.81.20.1051.239a-e1.0001.6140.164
68R. sulcatus_321.33b-n18.4624.11.4741.044h-q0.781.350.1661.085r-u0.8971.2290.091
69R. sulcatus_420.66f-t16.7224.292.0961.26ab0.881.750.2151.122j-u0.9661.3240.094
70R. sulcatus_521.42a-l17.7924.771.8551.098e-q11.30.1111.157d-t1.0311.4290.091
LSD0.0011.61 0.145 0.09
F-ANOVA15.41 *** 6.79 *** 6.70 ***
*** p < 0.001; in column means followed by the same letters are not significantly different.
Table
Table 5. Mean, minimal and maximal values as well as standard deviations (s.d.) for Le/P, d/E and Exp/P.
Table 5. Mean, minimal and maximal values as well as standard deviations (s.d.) for Le/P, d/E and Exp/P.
TraitLe/Pd/EExp/P
No.SpeciesMeanHomogeneous GroupsMinMaxs.d.MeanHomogeneous GroupsinMaxs.d.MeanHomogeneous GroupsMinMaxs.d.
1R. bifrons_10.832c-k0.7480.9280.0430.111t-x0.0440.1640.0310.042n-r0.0300.0630.007
2R. bifrons_20.841b-j0.7620.9220.0420.137h-w0.0910.2060.0310.043l-r0.0310.0580.007
3R. bifrons_30.860a-e0.8110.9410.0310.127n-x0.0760.1810.0280.044i-r0.0330.0660.009
4R. bifrons_40.851a-i0.6980.9490.0490.120r-x0.0680.2210.0370.044k-r0.0350.0530.005
5R. bifrons_50.841b-j0.7310.9330.0430.121r-x0.0730.1950.0300.039r0.0320.0510.005
6R. caesius_10.834b-k0.7430.9200.0440.129n-x0.0650.2340.0390.050b-k0.0400.0670.006
7R. caesius_20.856a-i0.8060.9090.0260.168b-h0.0980.2470.0430.044j-r0.0310.0670.010
8R. caesius_30.845b-j0.7180.9190.0490.165b-k0.1000.3290.0510.042n-r0.0340.0530.005
9R. caesius_40.822i-k0.7040.9010.0450.210A0.1480.2750.0390.042n-r0.0310.0620.007
10R. caesius_50.836b-k0.7580.8990.0350.184ab0.1210.2780.0330.045i-r0.0370.0580.005
11R. constrictus_10.841b-j0.7510.9550.0500.155b-q0.0840.2650.0500.045h-r0.0370.0580.005
12R. constrictus_20.840b-j0.7010.9070.0430.136i-w0.0680.2100.0410.048d-n0.0400.0630.007
13R. divaricatus_10.857a-h0.6850.9160.0460.097x0.0560.1510.0240.055a-d0.0370.0740.011
14R. divaricatus_20.843b-j0.6920.9180.0440.123q-x0.0770.2290.0340.049c-l0.0410.0570.005
15R. divaricatus_30.834b-k0.7130.9090.0450.109v-x0.0610.2100.0330.051b-i0.0430.0770.007
16R. divaricatus_40.837b-k0.7890.9020.0290.107wx0.0550.1790.0320.055a-e0.0360.0700.010
17R. gracilis_10.844b-j0.7640.9090.0410.140f-v0.0790.2710.0450.042m-r0.0300.0550.007
18R. gracilis_20.858a-g0.7691.0080.0490.125o-x0.0790.2370.0410.044i-r0.0330.0770.009
19R. gracilis_30.866ab0.7970.9470.0420.141f-v0.0620.2330.0430.043l-r0.0330.0530.005
20R. gracilis_40.848a-i0.8070.9090.0270.134k-w0.0770.2670.0390.049c-n0.0350.0730.009
21R. gracilis_50.833b-k0.7420.9050.0450.128n-x0.0770.2340.0390.046g-r0.0350.0590.006
22R. henrici-egonis_10.831d-k0.7380.8950.0390.121r-x0.0690.2060.0290.047f-q0.0370.0580.006
23R. henrici-egonis_20.854a-i0.7700.9290.0370.167b-j0.1020.2450.0450.047g-q0.0370.0680.007
24R. henrici-egonis_30.842b-j0.7710.9910.0480.113s-x0.0710.1560.0190.049c-n0.0360.0780.008
25R. henrici-egonis_40.844b-j0.7660.8980.0330.121r-x0.0530.2330.0450.057ab0.0330.0800.012
26R. henrici-egonis_50.848a-i0.7720.9140.0340.137h-w0.0680.2000.0310.044j-r0.0320.0550.006
27R. idaeus_10.866ab0.7960.9270.0390.170b-g0.1120.2470.0380.044i-r0.0350.0580.005
28R. idaeus_20.825f-k0.7270.8860.0370.181a-c0.1390.2280.0230.048e-o0.0370.0880.012
29R. idaeus_30.840b-j0.7110.9120.0380.158b-n0.1050.2420.0310.046g-r0.0330.0570.007
30R. idaeus_40.860a-e0.7500.9310.0410.171b-f0.1110.2210.0280.054a-f0.0370.0690.009
31R. idaeus_50.849a-i0.7660.9310.0400.171b-f0.1270.2260.0270.049c-n0.0380.0690.007
32R. nessensis_10.847b-j0.7440.9300.0460.134k-w0.0570.2680.0420.049c-m0.0340.0680.007
33R. nessensis_20.844b-j0.7480.9190.0430.152c-r0.0930.2580.0460.044k-r0.0330.0560.005
34R. nessensis_30.833b-k0.6790.9240.0460.124p-x0.0580.2260.0390.041o-r0.0280.0540.005
35R. nessensis_40.836b-k0.7590.9170.0350.144e-s0.0620.2440.0470.042m-r0.0270.0600.008
36R. nessensis_50.836b-k0.7540.9220.0380.125o-x0.0670.2070.0360.053a-g0.0340.0700.010
37R. opacus_10.864a-d0.8000.9310.0300.128n-x0.0970.1630.0180.047f-p0.0340.0610.007
38R. opacus_20.803k0.6710.8780.0470.164b-l0.1010.2770.0450.055a-c0.0370.0720.011
39R. plicatus_10.855a-i0.7740.8970.0300.123q-x0.0810.1930.0260.048e-p0.0380.0640.007
40R. plicatus_20.867ab0.7830.9460.0320.150c-r0.0810.2500.0380.059a0.0360.0990.019
41R. plicatus_30.814jk0.7000.9060.0460.145e-r0.0820.2980.0590.046g-q0.0210.0710.013
42R. plicatus_40.825g-k0.7550.8890.0330.157b-o0.1060.2550.0390.048d-n0.0210.0720.013
43R. plicatus_50.851a-i0.7710.9180.0390.167b-j0.0820.2630.0430.043l-r0.0270.0550.006
44R. praecox_10.853a-i0.7840.9380.0410.128n-x0.0570.2100.0390.051b-j0.0330.0660.011
45R. praecox_20.848a-i0.7780.9350.0390.173b-e0.1060.2400.0330.044i-r0.0330.0670.006
46R. praecox_30.859a-g0.7510.9050.0340.132l-w0.0930.2180.0280.045h-r0.0350.0570.005
47R. praecox_40.859a-f0.7530.9180.0350.136h-w0.0870.2050.0280.041p-r0.0340.0530.005
48R. radula_10.865a-c0.7880.9350.0310.110u-x0.0580.1700.0280.045h-r0.0270.0660.011
49R. radula_20.850a-i0.6960.9250.0470.128n-x0.0990.1780.0210.041p-r0.0320.0520.006
50R. radula_30.830e-k0.6990.9120.0480.143e-s0.0830.2830.0520.042n-r0.0350.0510.004
51R. radula_40.824h-k0.7600.8860.0260.156b-p0.1020.2330.0330.052b-h0.0330.0820.011
52R. radula_50.848a-j0.7500.9080.0390.098x0.0610.2230.0340.044j-r0.0370.0590.006
53R. saxatilis_10.881a0.8310.9390.0290.181a-c0.1190.2900.0440.040qr0.0310.0480.005
54R. saxatilis_20.829e-k0.7040.9980.0570.164b-l0.0870.2470.0400.048e-o0.0180.0680.012
55R. saxatilis_30.850a-i0.7750.9230.0340.161b-m0.1050.2500.0420.050c-l0.0310.0770.010
56R. saxatilis_40.847b-j0.7120.9020.0400.179a-d0.0930.2790.0440.045h-r0.0350.0580.005
57R. saxatilis_50.849a-i0.7540.9140.0370.168b-i0.1180.2310.0300.047g-q0.0360.0690.007
58R. scissus_10.844b-j0.7210.9600.0450.125o-x0.0800.1750.0290.046h-r0.0320.0590.007
59R. scissus_20.850a-i0.7530.9410.0390.164b-l0.0770.3050.0560.047f-p0.0380.0570.005
60R. scissus_30.847b-j0.7740.8940.0320.169b-g0.0890.2500.0470.044i-r0.0350.0590.005
61R. sprengelii_10.853a-i0.7800.9400.0350.152b-r0.0930.2500.0360.049c-m0.0340.0640.009
62R. sprengelii_20.840b-j0.6950.9280.0480.135j-w0.0840.2300.0350.042m-r0.0310.0520.005
63R. sprengelii_30.844b-j0.7650.9170.0380.136i-w0.0840.2530.0390.047g-q0.0370.0590.006
64R. sprengelii_40.847a-j0.7340.9310.0380.143e-t0.1000.2200.0290.041o-r0.0280.0580.007
65R. sprengelii_50.867ab0.8060.9150.0300.138g-w0.0950.2550.0380.042n-r0.0330.0550.005
66R. sulcatus_10.840b-j0.7510.9170.0410.139g-w0.0850.2270.0350.046h-r0.0310.0640.008
67R. sulcatus_20.859a-f0.7980.9160.0310.148d-r0.1040.2220.0300.045h-r0.0380.0560.005
68R. sulcatus_30.846b-j0.7310.9170.0430.131m-w0.0680.2200.0370.046h-r0.0320.0650.009
69R. sulcatus_40.839b-j0.7690.9050.0370.141f-u0.0770.2420.0430.055a-d0.0380.0920.012
70R. sulcatus_50.867ab0.7860.9350.0340.128n-x0.0720.2100.0360.045h-r0.0360.0560.006
LSD0.0010.034 0.032 0.007
F-ANOVA3.51 *** 10.96 *** 8.64 ***
*** p < 0.001; in the column means followed by the same letters are not significantly different.
Table
Table 6. Exine ornamentation types and subtypes of Rubus species studied (according to Ueda and Tomita [40] classification).
Table 6. Exine ornamentation types and subtypes of Rubus species studied (according to Ueda and Tomita [40] classification).
Striate Exine Ornamentation Type or SubtypeSpecies
IIAR. divaricatus, R. gracilis, R. henrici-egonis, R. plicatus, R. praecox, R. radula, R. scissus,
R. sprengelii
IIBR. caesius, R. idaeus
IIIAR. bifrons, R. nessensis, R. opacus, R. saxatilis
IIIBR. constrictus, R. sulcatus
Table
Table 7. Correlation coefficients between the first two canonical variables and original traits.
Table 7. Correlation coefficients between the first two canonical variables and original traits.
TraitFirst Canonical VariateSecond Canonical Variate
P−0.951 ***−0.249 *
Le−0.914 ***−0.206
d−0.485 ***0.804 ***
E−0.918 ***0.189
Exp−0.062−0.384 **
P/E0.141−0.625 ***
Le/P0.0860.162
d/E−0.2010.861 ***
Exp/P0.585 ***−0.161
Percentage of explained multivariate variability33.9824.42
* p < 0.05; ** p < 0.01; *** p < 0.001.
Table
Table 8. The ranking of features determining intraspecific variability.
Table 8. The ranking of features determining intraspecific variability.
SpeciesRanking
1
(the Largest)		23456789
(the Smallest)
R. bifronsExp ***Le ***P **d **d/E *Exp/P *Le/PEP/E
R. caesiusE ***P ***d ***Le ***d/E ***P/E ***Exp/P ***Le/P *Exp
R. constrictusd *Exp/P *E *Exp *d/EP/EPLeLe/P
R. divaricatusE ***Exp ***P/E ***Le **P *d/E *Exp/P *Le/Pd
R. gracilisE ***P ***Le ***P/E ***Exp/P *d *Le/P *Expd/E
R. henrici-egonisd ***E ***Exp/P ***d/E ***P ***Le ***Exp ***P/E ***Le/P
R. idaeusP ***E ***d ***Exp ***Exp/P ***Le/P ***Le **P/E *d/E
R. nessensisExp/P ***Exp ***P ***E **Le *d *d/EP/ELe/P
R. opacusP ***Le/P ***Exp ***d ***E ***d/E ***Exp/P **LeP/E
R. plicatusP ***Le/P ***E ***Exp/P ***P/E ***d ***d/E **Exp **Le *
R. praecoxP ***Le ***Exp ***d/E ***Exp/P ***E ***d **P/ELe/P
R. radulad/E ***P ***Le ***Exp ***d ***Exp/P ***Le/P ***E **P/E
R. saxatilisLe ***P ***Le/P ***Exp/P ***E ***P/E ***Exp ***d/Ed
R. scissusd ***d/E ***P/E ***P ***Le **ExpExp/PELe/P
R. sprengeliiExp ***Exp/P ***P/E ***P **Le *Le/PdEd/E
R. sulcatusE ***Exp ***Exp/P ***P/E ***P ***Le ***Le/P *d/Ed
* p < 0.05; ** p < 0.01; *** p < 0.001.
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Lechowicz, K.; Bocianowski, J.; Wrońska-Pilarek, D. Pollen Morphological Inter- and Intraspecific Variability in Selected Species of Rubus L. (Rosaceae). Forests 2022, 13, 1946. https://doi.org/10.3390/f13111946

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Lechowicz K, Bocianowski J, Wrońska-Pilarek D. Pollen Morphological Inter- and Intraspecific Variability in Selected Species of Rubus L. (Rosaceae). Forests. 2022; 13(11):1946. https://doi.org/10.3390/f13111946

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Lechowicz, Kacper, Jan Bocianowski, and Dorota Wrońska-Pilarek. 2022. "Pollen Morphological Inter- and Intraspecific Variability in Selected Species of Rubus L. (Rosaceae)" Forests 13, no. 11: 1946. https://doi.org/10.3390/f13111946

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